1. Field of the Invention
This invention relates to apparatus used to separate molecules according to size. More specifically, this invention relates to the separation of molecules by electrophoresis.
While the present invention is described herein with reference to a particular embodiment for a particular application, it is understood that the invention is not limited thereto. Those having ordinary skill in the art and access to the teachings provided herein will recognize additional embodiments within the scope thereof.
2. Description of the Related Art
The conventional process of electrophoresis has heretofore been used, with limited success, in the separation and size measurement of molecules. The application of gel electrophoresis to DNA molecules has proven useful in sequence analysis, restriction mapping and physical mapping of DNA.
Current methods of electrophoresis typically utilize an electric field to separate charged molecules embedded within a gel matrix. The molecules are separated, within the gel, primarily on the basis of size, charge and physical conformation.
Conventional electrophoresis is performed by an apparatus which generates a unidirectional, uniform electric field provided by a single pair of electrodes. The field produced by such systems is static in that it is time invariant. The charged molecules move within the gel in response to the static field. The mobility of each molecule is related to the logarithm of its molecular weight and the concentration of the gel. Consequently, useful separations of molecules of various sizes have tended to require gels of differing concentrations. Further, insignificant differences in mobility among large macromolecules e.g. DNA molecules in excess of 50,000 base pairs (50 kb) have limited the utility of conventional static field electrophoresis methods as separation tools. That is, prior systems offer limited resolution with respect to large molecules.
The introduction of electrode configurations which generate alternating electric fields transverse to the net direction of macromolecular migration has enabled separation of DNA molecules larger than 50 kb. As disclosed in U.S. Pat No. 4,473,452, issued Sept. 25, 1984 to Schwartz and Cantor, a technique commonly known as pulsed field gradient gel electrophoresis (PFG) employs uniform or non-uniform alternately pulsed electric fields oriented in a predetermined manner to separate molecules.
In "Cell", Vol. 37, May 1984, pp. 67-75, Schwartz and Cantor describe PFG separation of DNA molecules up to 2000 kb, noting that non-uniform electric fields are critical in achieving high resolution. Resolution of DNA fragments separated by PFG is typically a function of the pulse time, geometry and strength of applied electric fields. However, point electrode elements typically utilized in conventional PFG devices are often capable of assuming only a pair of electric potentials thus constraining the range of fields which may be applied to a gel.
Though PFG electrophoresis enables separation of larger DNA macromolecules than conventional static field approaches, the qualitative usefulness of PFG separations generated by non-uniform electric fields has been limited by the production of curved trajectory migrations. That is, as a result of the application of typically non-uniform, spatially nonhomogenous electric fields in PFG electrophoresis, DNA samples will typically follow nonlinear paths. As is known in the art, comparisons between the migrations of known and unknown samples are typically more accurate when the trajectories of both samples are linear rather than nonlinear.
In "Analytical Biochemistry", Vol. 156, 1986, pp. 274-285, McPeek describes a technique of using alternately pulsed, opposing nonhomogenous fields to counteract the effect of "lane bending" (curved migration trajectories). This approach improved linearity in the electrophoretic migration path, but the conventional bistable point electrode configurations utilized allow only limited control of electric fields.
An alternative approach developed to achieve improved linearity in electrophoretic migration is known as field inversion (FI) gel electrophoresis. In FI electrophoresis the applied electric field is periodically inverted for unequal intervals using a pair of conventional electrodes with net migration of a sample occurring in the field direction associated with the longer interval. The opposing electric fields in FI electrophoresis are said to have a "reorientation angle" of 180 degrees while the reorientation angle in conventional PFG electrophoresis is typically between 90 to 150 degrees. With periodic switching between electric fields, FI electrophoresis has produced separation of DNA in the 10 kb to 1600 kb size range. Resolution of DNA up to 750 kb has been better in FI electrophoresis than in conventional PFG electrophoresis.
Despite offering improved resolution of certain DNA size ranges relative to PFG electrophoresis, conventional FI electrophoresis typically does not induce a monotonic relationship between migration mobility and molecular weight. Specifically, in FI electrophoresis molecules differing in size by several times may exhibit similar net migration in the direction of separation. In some instances this limitation has been partially overcome by linearly increasing/decreasing the duration of the applied field pulses as a function of time (switch time ramping).
Electrophoretic separation by contour-clamped homogenous electric fields (CHEF) has recently been utilized to separate DNA in excess of 2000 kb with generally improved resolution relative to PFG or FI electrophoresis. See "Science", Vol. 234, Dec. 19, 1986, pp. 1582-1585 by Chu et al. The apparatus used includes multiple electrodes evenly spaced along a polygonal contour. Electrodes on opposite sides of the polygon may be utilized to define the electric field orientation within an electrophoretic gel. Electrodes on the remaining sides of the polygon are linked by equal resistors and hence have associated intermediate potentials. These intermediate potentials are equivalent to the potentials which would exist at the position occupied by the intermediate electrode in a completely homogenous field. The field is typically periodically switched between two pairs of electrodes on opposing sides of the polygon with the potentials of the intermediate electrodes on remaining sides of the polygon also adjusted accordingly. Field homogeneity within the gel allows expedient comparison between samples in different regions of a single gel as each sample experiences a substantially equivalent field.
While this technique provides more effective separation of certain large DNA macromolecules than that afforded by conventional PFG or FI electrophoretic techniques, the particular CHEF electrophoresis scheme proposed by Chu et al. has two primary limitations. First, the electric field reorientation angle is effectively defined by, and restricted to, the geometry of the electrode configuration. For example, as noted by Chu on page 1583 of the above-noted reference, a square electrode configuration produces a 90 degree reorientation angle while a hexagonal electrode configuration may generate reorientation angles of either 60 or 120 degrees. This constraint on reorientation angles may limit the utility of conventional CHEF electrophoresis. Hence, the reorientation angles or combinations thereof necessary for optimum separation of DNA within given size ranges may be unattainable with the apparatus and teaching of Chu.
Second, the system of Chu is generally limited to the production of a fixed magnitude field during the electrophoretic process. This constant field magnitude can be disadvantageous as utilizing electric fields with magnitude gradients may aid in separation of desired DNA size ranges.
As a result of the above limitations, conventional CHEF electrophoretic systems are typically capable of generating only two "field states". That is, the electric fields produced during CHEF electrophoresis are of fixed magnitude and are usually constrained to alternate in two directions. Thus, potentially improved methods of electrophoretic separation requiring generation of more than a pair of field states could generally not be implemented using conventional CHEF systems.
Hence a need in the art exists for an electrophoretic apparatus for separating particles within a medium which is capable of generating electric fields having adjustable magnitudes and orientations.